Microscope and segmenting device for a microscope

ABSTRACT

A microscope, in particular for confocal scanning microscopy, having a light source ( 1 ) for illuminating an object ( 6 ) to be investigated. An optical device ( 9, 12 ) is provided for splitting transmitted light ( 15 ) passing through the object ( 6 ) and fluorescent light ( 10, 13 ) produced in the object ( 6 ). A segmenting device ( 17 ) acting on the transmitted light ( 15 ) is configured with regard to a detection-light path which is as short as possible in such a way that the segmenting device ( 17 ) is arranged between the object ( 6 ) and the light-splitting device ( 9, 12 ).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of a German patent applicationDE P 100 03 762.3 filed Jan. 28, 2000 which is incorporated by referenceherein.

FIELD OF THE INVENTION

[0002] The invention relates to a microscope. In particular theinvention relates to a confocal scanning microscopy having a segmentingdevice.

[0003] Furthermore, the invention relates to a segmenting device whichis used, in particular, in the abovenamed microscope or confocalmicroscope.

BACKGROUND OF THE INVENTION

[0004] A microscope generally comprises a light source and a focussingoptical system with the aid of which the light from the source isfocussed onto a pinhole stop. In this case, a beam splitter, a scanningdevice for beam control, a microscope optical system, a detection stopand detectors for detecting detection and/or fluorescent light areprovided.

[0005] In a confocal microscope, the illuminating light is mostlycoupled in via the beam splitter. The focus of the light beam is movedwith the aid of the scanning device in a sample plane. For this purpose,it is customary to use two mirrors which are tilted, the deflection axesmostly being perpendicular to one another, such that one mirror deflectsin the X-direction and the other deflects in the Y-direction.

[0006] It is also possible in a transmitted-light arrangement for thefluorescent light or the transmitted light—the transmission of theexciting light—, for example, to be detected on the condenser side, thatis to say on the side of a condenser arranged downstream of the object.The detection-light beam then does not pass via the scanning mirrors tothe detector. Such an arrangement is denoted as a non-descanningarrangement.

[0007] In order to detect the fluorescent light, there would be a needin the transmitted-light arrangement for a condenser-side detection stopin order—as in the descanning arrangement described—to achieve athree-dimensional resolution. In the case of two-photon excitation,however, it is possible to dispense with a condenser-side detectionstop, since the probability of excitation is a function of the square ofthe photon density or the intensity, which is naturally much higher atthe focus than in the neighbouring regions. The fluorescent light to bedetected therefore originates with high probability in overwhelmingproportion from the focussing region, and this renders superfluousfurther differentiation of fluorescence photons from the focussingregion from fluorescence photons from the neighbouring regions with theaid of a stop arrangement.

[0008] Particularly against the background of a yield of fluorescencephotons which is low in any case for two-photon excitation, anon-descanning arrangement in which less light is generally lost on thedetection-light path is of interest. Nevertheless, because of the lackof marking in living preparations, cell contours, for example, alsocannot be detected sufficiently well in the case of this type ofobservation of fluorescent light, and so it would be desirable to besimultaneously able to observe the transmitted light, which would permitclear conclusions.

[0009] A microscope for simultaneously detecting fluorescent andtransmitted light is already known to the applicant from an earlierpatent application. The known microscope comprises downstream of acondenser a light-splitting device in the form of at least one colourbeam splitter which spatially separates or splits the fluorescent lightfrom the transmitted light.

[0010] For the purpose of transmitted-light contrast microscopy, in thecase of the known microscope the segmenting device required for contrastis arranged in the form of a segment stop in a Fourier plane of thetransmitted light downstream of the light-splitting device or downstreamof the colour beam splitter. This requires a long detection-light pathdownstream of the light-splitting device, which path also has to be setup in addition to the standard microscope equipment.

BRIEF SUMMARY OF THE INVENTION

[0011] It is therefore the object of the present invention to specify amicroscope having a detection-light path which is as short as possibleand can be implemented with the aid of structurally simple means.

[0012] The above object is achieved by a microscope which comprises: alight source for illuminating an object to be investigated, a an opticaldevice for splitting transmitted light passing through the object andfluorescent light generated in the object, a segmenting device acting onthe transmitted light, wherein the segmenting device is arranged betweenthe object and the optical device.

[0013] It is a further object of the present invention to specify aconfocal microscope having a detection-light path which is as short aspossible and can be implemented with the aid of structurally simplemeans.

[0014] The above object is achieved by a confocal microscope whichcomprises: a laser light source for illuminating an object to beinvestigated, a an optical device for splitting transmitted lightpassing through the object and fluorescent light generated in theobject, a segmenting device acting on the transmitted light, wherein thesegmenting device is arranged between the object and the optical device.

[0015] It is an additional object of the invention to provide asegmenting device which allows a detection-light path to be as short aspossible and simple to implement.

[0016] The above object is achieved by a segmenting device whichcomprises: a transparent substrate and a colour-selective coating formedon said transparent substrate.

[0017] The advantage of the invention is, that an optimized arrangementof the segmenting device solves the above object in a surprisinglysimple way. For this purpose, the segmenting device is no longerarranged downstream of the light-splitting device, but between theobject and the light-splitting device. A detection-light path, alreadyprescribed by the arrangement of the light-splitting device, between theobject and the light-splitting device is employed in this case, inaddition, by virtue of the arrangement of the segmenting device on thisdetection-light path. The creation of an additional detection-light pathis thereby avoided.

[0018] Consequently, the microscope according to the invention has adetection-light path which is as short as possible with the aid ofstructurally simple means.

[0019] In a concrete embodiment of the microscope, a condenser for thetransmitted light and the fluorescent light could be arranged on theside of the object averted from the light source. The segmenting devicecould then be arranged in a simple way between the condenser and theoptical device, in which case with regard to segmenting which is aseffective as possible, it is advantageous to arrange the segmentingdevice in a Fourier plane between the condenser and the optical device.In any case, the segmenting device could act exclusively on thetransmitted light, in which case the fluorescent light can pass throughthe segmenting device without being influenced.

[0020] The device for inserting optical components into a beam pathcould have a revolver mechanism or a sliding magazine in order to ensurea particularly reliable insertion of the optical components into thebeam path. Not only a very reliable, but also a very simple insertion ofa segmenting device into the beam path is achieved thereby.

[0021] In a particularly versatile way, different segmenting devicescould be arranged in the device. Depending on the required investigationmethods, it is therefore possible to insert the suitable segmentingdevice into the beam path.

[0022] The most varied components come into consideration as segmentingdevice. In particular, the segmenting device could have a segment stop,a segment phase stop or a segment phase filter.

[0023] In a structurally particularly simple configuration, thesegmenting device could have a transparent substrate with acolour-selective coating. This implements not only a particularly simplesegmenting device, but also an exceptionally thin segmenting devicewhich can be inserted without difficulty into a microscope of the typementioned at the beginning. The coating could in this case be opaque tothe transmitted light and transparent to the fluorescent light.

[0024] The coating could advantageously be a dielectric coating. In anycase, in a particularly suitable way, the coating could bevapour-deposited onto the substrate. Reliable adhesion of the coating onthe substrate can thereby be achieved.

[0025] In an alternative refinement, the segmenting device could have asuitably configured colour filter. The colour filter could, inparticular, be tailored in accordance with a known segment stop. Variousmaterials come into consideration as colour filter material. In thiscase, a configuration of the colour filter from plastic, preferablyplastic film, is particularly easy to handle. However, it is alsoconceivable for the colour filter to be configured from glass.

[0026] In a further alternative refinement, the segmenting device couldhave a colour LCD matrix. Such a colour LCD matrix permits variation ofthe segment or stop shape and variation of the colour properties withoutfurther redesigning.

[0027] In the simplest case, the optically active part of the segmentingdevice could be arranged perpendicular to an optical axis of themicroscope. In a conventional segment stop, the optically active part isformed by an essentially flat region. In the case, for example, of acoated substrate, the coating would be the optically active part of thesegmenting device.

[0028] In order to avoid reflections which could run back into the lightsource, the optically active part of the segmenting device could bearranged in a fashion deviating from the perpendicular to an opticalaxis of the microscope. In this case, the aim is, in particular, aslight deviation which does not influence the optical action of thesegmenting device.

[0029] The segmenting device or the optically active part of thesegmenting device can have different geometrical shapes, depending onthe application. In a particularly simple way, the optical device isconfigured as a light-splitting device and could have at least onecolour beam splitter. In this case a plurality of colour beam splitterscould, specifically, be arranged in series to enable differentwavelengths or wavelength regions to be split.

[0030] As an alternative to this, the light-splitting device could haveat least one partially transparent mirror. A bandpass or blocking filtercould be arranged downstream of this mirror or these mirrors. In thecase of the use of mirrors as splitting component as well, a pluralityof such mirrors could be arranged in series, if appropriate with adownstream bandpass or blocking filter. It is also possible thereby tosplit the fluorescent light into a plurality of spectral regions.

[0031] It is possible as an alternative to the use of colour beamsplitters or mirrors to make use for splitting of a multiband detectorwhich is described, for example, in DE 199 02 625 A1. Such a multibanddetector can also be used to split the fluorescent light into aplurality of spectral regions.

[0032] With regard to reliable detection of the transmitted light andthe fluorescent light, it would be possible to arrange at least onefluorescent-light detector for detecting the fluorescent light and atleast one transmitted-light detector for detecting the transmittedlight, for the purpose of simultaneously detecting fluorescent andtransmitted light, on the side of the object averted from the lightsource. In a particularly compact refinement of the microscope, thefluorescent light and the transmitted light could be detected in thesame detector. With regard to as clear a differentiation as possible,the fluorescent light and the transmitted light could, however, also bedetected in different detectors.

[0033] A laser could be used in a particularly advantageous way as lightsource. However, it is also conceivable to use other suitable lightsources.

[0034] The segmenting device could, for example, operate using the Dodtprinciple or using the Hoffinan principle. In this case, the segmentingdevice does not —as is usual—comprise a solid opaque segment stop. Inthe case of a segmenting device designed as a coated substrate, thecoating is transparent to the fluorescent light, while it is opaque tothe transmitted light—two—or multiphoton exciting light.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0035] There are various possibilities of configuring and developing theteaching of the present invention in an advantageous way. For thispurpose, reference is made, to the following description of preferredexemplary embodiments of the invention. In the drawing:

[0036]FIG. 1 shows a diagrammatic illustration of an exemplaryembodiment of a microscope according to the invention, and

[0037]FIG. 2 shows a diagrammatic illustration of an exemplaryembodiment of a segmenting device according to the invention with asuitably coated substrate.

DETAILED DESCRIPTION OF THE INVENTION

[0038]FIG. 1 shows an exemplary embodiment of a microscope according tothe invention in a diagrammatic illustration. The microscope is aconfocal laser scanning microscope. The microscope has a light source 1designed as a laser. The light source 1 emits an illuminating light beam2 which is reflected to a scanning device 4 via a main beam splitter 3.The scanning device 4 leads the illuminating light beam 2 through amicroscope optical system or an objective 5 via an object 6. Both thetransmitted light passing through the object 6 and the fluorescent lightproduced in the object 6 reach via a condenser 7 and a deflecting mirror8 a first colour beam splitter 9 which splits the spectrally lower-waveregion 10 of the fluorescent light and reflects it to afluorescent-light detector 11. Via a colour beam splitter 12, thespectrally higher-wave region 13 of the fluorescent light is reflectedto a further fluorescent-light detector 14. The transmitted light 15reaches a transmitted-light detector 16 arranged in the straight aheaddirection.

[0039] The microscope consequently has a light source 1 for illuminatingan object 6 to be investigated. Furthermore, the microscope has anoptical device which is configures as a light-splitting device with twocolour beam splitters 9 and 12 for splitting transmitted light 15passing through the object 6 and fluorescent light 10 and 13 produced inthe object. Finally, the microscope comprises a segmenting device, inthe form of a colour segment stop 17, which acts on the transmittedlight 15. With regard to a detection-light path which is as short aspossible, the segmenting device is arranged between the object 6 or thecondenser 7 and the light-splitting device. The microscope is suitablefor transmitted-light contrast microscopy on account of the segmentingdevice.

[0040] The microscope shown in FIG. 1 further has a detector 18 which isarranged on the objective side and is normally not used intransmitted-light contrast microscopy.

[0041] Both fluorescent-light detectors 11 and 14 are arranged on theside of the object 6 averted from the light source 1. Furthermore, thetransmitted-light detector 16 is arranged on the side of the object 6averted from the light source 1.

[0042] The segmenting device in the form of the colour segment stop 17is arranged in a Fourier plane between the condenser 7 and thelight-splitting device, more precisely the deflecting mirror 8. Arevolver mechanism or a sliding magazine can be used to insert thecolour segment stop 17.

[0043]FIG. 2 shows a diagrammatic representation of an exemplaryembodiment of a segmenting device according to the invention. Thesegmenting device has a flat substrate 19 with a coating 20. The coating20 is preferably dielectric and vapour-deposited onto the substrate 19.The coating 20 vapour-deposited onto the transparent substrate is opaqueto the transmitted light and transparent to the fluorescent light. Inparticular, the dielectric coating 20 is transparent to light ofwavelengths which are less than 790 nm. Light of higher wavelengths isreflected. The wavelength limit with regard to the transmission andreflection of light can, however, also be at another suitablewavelength.

[0044] With regard to further advantageous refinements and developmentsof the microscope according to the invention, and to the segmentingdevice according to the invention, reference may be made for the purposeof avoiding repetition to the general part of the description and to theattached patent claims.

[0045] Finally, it may be pointed out expressly that the above-describedexemplary embodiments of the microscope according to the invention andof the segmenting device according to the invention serve merely todiscuss the teaching claimed but do not limit the latter to theseexemplary embodiments.

PARTS LIST

[0046]1 Light source

[0047]2 Illuminating light beam

[0048]3 Main beam splitter

[0049]4 Scanning device

[0050]5 Objective

[0051]6 Object

[0052]7 Condenser

[0053]8 Deflecting mirror

[0054]9 Colour beam splitter

[0055]10 Wavelength region

[0056]11 Fluorescent-light detector

[0057]12 Colour beam splitter

[0058]13 Wavelength region

[0059]14 Fluorescent-light detector

[0060]15 Transmitted-light beam

[0061]16 Transmitted-light detector

[0062]17 Colour segment stop

[0063]18 Detector

[0064]19 Substrate

[0065]20 Coating

What is claimed is:
 1. A microscope comprising: a light source (1) forilluminating an object (6) to be investigated, a an optical device (9,12) for splitting transmitted light (15) passing through the object (6)and fluorescent light (10, 13) generated in the object (6), a segmentingdevice (17) acting on the transmitted light (15), wherein the segmentingdevice (17) is arranged between the object (6) and the optical device(9, 12).
 2. Microscope according to claim 1 , characterized in that acondenser (7) for the transmitted light (15) and the fluorescent light(10, 13) is arranged on the side of the object (6) averted from thelight source (1).
 3. Microscope according to claim 2 , characterized inthat the segmenting device (17) is arranged between the condenser (7)and the optical device (9, 12).
 4. Microscope according to claim 3 ,characterized in that the segmenting device (17) is arranged in aFourier plane between the condenser (7) and the optical device (9, 12).5. Microscope according to claim 1 , characterized in that thesegmenting device (17) is arranged on a device for inserting opticalcomponents into a beam path.
 6. Microscope according to claim 1 ,characterized in that the segmenting device (17) has a segment stop, asegment phase stop or a segment phase filter.
 7. Microscope according toclaim 1 , characterized in that the segmenting device (17) has atransparent substrate (19) with a colour-selective coating (20). 8.Microscope according to claim 7 , characterized in that the coating (20)is opaque to the transmitted light (15) and transparent to thefluorescent light (10, 13).
 9. Microscope according to claim 1 ,characterized in that the segmenting device has a colour LCD matrix. 10.Microscope according to claim 1 , characterized in that the opticallyactive part of the segmenting device (17) is arranged perpendicular toan optical axis of the microscope.
 11. A confocal microscope comprising:a laser light source (1) for illuminating an object (6) to beinvestigated, a an optical device (9, 12) for splitting transmittedlight (15) passing through the object (6) and fluorescent light (10, 13)generated in the object (6), a segmenting device (17) acting on thetransmitted light (15), wherein the segmenting device (17) is arrangedbetween the object (6) and the optical device (9, 12).
 12. The confocalmicroscope according to claim 11 , characterized in that a condenser (7)for the transmitted light (15) and the fluorescent light (10, 13) isarranged on the side of the object (6) averted from the light source(1).
 13. The confocal microscope according to claim 12 , characterizedin that the segmenting device (17) is arranged between the condenser (7)and the optical device (9, 12).
 14. The confocal microscope according toclaim 11 , characterized in that the segmenting device (17) is arrangedon a device for inserting optical components into a beam path.
 15. Theconfocal microscope according to claim 11 , characterized in that thesegmenting device (17) has a transparent substrate (19) with acolour-selective coating (20).
 16. The confocal microscope according toclaim 15 , characterized in that the coating (20) is opaque to thetransmitted light (15) and transparent to the fluorescent light (10,13).
 17. The confocal microscope according to claim 11 characterized inthat a multiband detector arranged to the optical device (9, 12). 18.The confocal microscope according to claim 11 , characterized in that atleast one fluorescent light detector (11, 14) for detecting thefluorescent light (10, 13) and at least one transmitted-light detector(16) for detecting the transmitted light (15) are arranged, for thepurpose of simultaneously detecting fluorescent and transmitted light(10, 13; 15), on the side of the object (6) averted from the lightsource (1).
 19. A segmenting device (17) comprising: a transparentsubstrate (19) and a colour-selective coating (20) formed on saidtransparent substrate (19).
 20. The segmenting device according to claim19 , characterized in that the coating (20) is opaque to the transmittedlight (15) and transparent to the fluorescent light (10, 13). 21.Segmenting device according to claim 19 , characterized in that thecoating (20) is dielectric.
 22. Segmenting device according to claim 19, characterized in that the coating (20) is vapour-deposited onto thesubstrate (19).
 23. Segmenting device, according to claim 19 ,characterized in that the segmenting device has a suitably configured,preferably tailored, colour filter.
 24. Segmenting device according toclaim 23 , characterized in that the colour filter is produced fromplastic.
 25. Segmenting device, according to claim 19 , characterized inthat the segmenting device has a colour LCD matrix.